Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos.

Abstract

Abstract. Using calcium-sensitive dyes together with their dextran conjugates and confocal microscopy, we have looked for evidence of localized calcium signaling in the region of the nucleus before entry into mitosis, using the sea urchin egg first mitotic cell cycle as a model. Global calcium transients that appear to originate from the nuclear area are often observed just before nuclear envelope breakdown (NEB). In the absence of global increases in calcium, confocal microscopy using Calcium Green-1 dextran indicator dye revealed localized calcium transients in the perinuclear region.We have also used a photoinactivatable calcium chelator, nitrophenyl EGTA (NP-EGTA), to test whether the chelator-induced block of mitosis entry can be reversed after inactivation of the chelator. Cells arrested before NEB by injection of NP-EGTA resume the cell cycle after flash photolysis of the chelator. Photolysis of chelator triggers calcium release. Treatment with caffeine to enhance calcium-induced calcium release increases the amplitude of NEB-associated calcium transients. These results indicate that calcium increases local to the nucleus are required to trigger entry into mitosis. Local calcium transients arise in the perinuclear region and can spread from this region into the cytoplasm. Thus, cell cycle calcium signals are generated by the perinuclear mitotic machinery in early sea urchin embryos. INTRACELLULAR calcium is now known to perform a wide variety of functions as a second messenger. It is implicated in the control of exocytosis, protein synthesis, locomotion, and synaptic transmission (for reviews see Campbell, 1983;Berridge, 1993; Brostrom and Brostrom, 1990;Rasmussen and Rasmussen, 1990). It is increasingly clear that calcium signals can originate in specific cytoplasmic domains (Lechleiter et al., 1991;Rizzuto et al., 1993;Meyer et al., 1995;Petersen, 1995;Kasai, 1995). A body of evidence also points to a role for calcium ions as one of the mechanisms enabling passage through cell cycle control points (for reviews see Whitaker and Patel, 1990;Steinhardt, 1990;Lu and Means, 1993;Whitaker, 1995).The evidence for a role of calcium ions during mitosis comes from observations that embrace many different aspects of cell cycle physiology. For example, many components of the intracellular calcium signaling system localize to the area of the nucleus just before mitosis entry and to the mitotic apparatus during mitosis (Silver et al., 1980; Keihart, 1981;Petzelt, 1979Petzelt, , 1984Petzelt and Hafner, 1986;Wolniak et al., 1981; Paweletz and Firze, 1981;Hepler, 1980; Wolniak, 1983, 1984 as EGTA and BAPTA blocks both entry into, and exit from, mitosis (Steinhardt and Alderton, 1988;Twigg et al., 1988;Whitaker and Patel, 1990;Patel et al., 1989Patel et al., , 1990Ohsumi and Anraku, 1983;Iida et al., 1990; Lindsay et al., 1995). Entry into mitosis can also be induced prematurely by microinjection of calcium during a defined period before mitosis entry (Steinhardt and Alderton, 1988;Twigg et al., 1988;Patel et al., 1989...

An increase in [Ca2+]i is sufficient but not necessary for driving mitosis in early mouse embryos

“…This idea was supported by early reports that [Ca 2+ ] is elevated at spindle poles before anaphase in PTK and endosperm cells (Keith et al, 1985;Ratan et al, 1986). More recently, using confocal imaging and ratiometric Ca 2+ indicators, localized Ca 2+ increases were detected in the perinuclear region just before NEBD in sea-urchin embryos (Wilding et al, 1996). Consistent with the ability to generate local Ca 2+ signals is the observation that, shortly before NEBD, the endoplasmic reticulum aggregates around the nucleus (Terasaki, 2000).…”

“…This was the case in fertilized embryos that generate Ca 2+ transients and in parthenogenetic embryos that do not (Tombes et al, 1992;Kono et al, 1996) (present study). Similarly, in sea-urchin embryos and fibroblasts, BAPTA inhibits mitosis despite the absence of detectable Ca 2+ transients in many cases (Kao et al, 1990;Wilding et al, 1996). These inconsistencies could be reconciled after it was found that local perinuclear [Ca 2+ ] increases, which were not detectable using epifluorescence microscopy, were detectable using ratiometric confocal microscopy in sea-urchin embryos (Wilding et al, 1996).…”

“…However, these epifluorescence studies cannot exclude the possibility that mitosis entry is triggered by localized [Ca 2+ ] elevations, such as those detected before NEBD in sea-urchin embryos (Wilding et al, 1996). Such localized Ca 2+ changes might also drive mitosis entry in parthenogenetic mouse embryos, which do not generate global Ca 2+ transients at NEBD.…”

“…Ca 2+ chelators such as EGTA and BAPTA prevent Ca 2+ release and block NEBD (Steinhardt and Alderton, 1988;Twigg et al, 1988). Conversely, NEBD can be induced by treatments that increase [Ca 2+ ] i (Wilding et al, 1996;Twigg et al, 1988;Steinhardt and Alderton, 1988). Similar findings have been made at the metaphase-anaphase transition, which can be reversibly inhibited by Ca 2+ buffers or by inhibiting Ins(1,4,5)P 3 receptors (InsP 3 Rs) .…”

Calcium signalling in early embryos

“…Microinjection of calcium or InsP 3 induces NEB precociously (Steinhardt & Alderton 1988;Twigg et al 1988) and calcium chelators block NEB reversibly (Wilding et al 1996). NEB in starfish embryos is also preceded by a calcium spike and blocked by heparin injection (Stricker 1995).…”